Identification of Residues within Two Regions Involved in Self-association of Viral Histone-like Protein p6 from Phage Ø29

Abril, Ana M.; Salas, Margarita; Hermoso, José Miguel

doi:10.1074/jbc.m002739200

Cited by 6 publications

(12 citation statements)

References 39 publications

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“…Thus, the capability of p6NÁ5 and p6NÁ13 deletion mutants to selfassociate was tested in vitro by glutaraldehyde crosslinking. Dimer formation was highly impaired in p6NÁ5, and undetectable in p6NÁ13 (Abril et al, 2000). Therefore, the observed self-association deficiency explains the DNA binding impairment of p6NÁ5 and p6NÁ13, suggesting that the N-terminal region, in addition to DNA binding, is involved in dimer formation.…”

Section: Protein P6 Mutants Deficient In Self-associationmentioning

confidence: 91%

“…Mutant p6A44V, located at the central part of the protein, showed impaired dimer formation ability, and a reduced capacity to bind DNA and to activate the initiation of Ø29 DNA replication. More dramatic was the phenotype of mutant p6I8T, which had a self-association capacity reduced at least 10-fold and did not bind DNA nor activate Ø29 DNA initiation of replication (Abril et al, 2000).…”

Section: Protein P6 Mutants Deficient In Self-associationmentioning

confidence: 99%

“…Protein p6 strongly stimulates the initiation (Blanco et al, 1986;Pastrana et al, 1985) as well as the transition to elongation (Blanco et al, 1988) of Ø29 DNA replication in an in vitro system with purified proteins. This stimulation required the formation of the protein p6 complex with Ø29 DNA terminal fragments (Serrano et al, 1989), and protein p6 mutants that do not bind DNA do not activate initiation of replication (Abril et al, 2000;Freire et al, 1994Freire et al, , 1996Otero et al, 1990;Otero and Salas, 1989).…”

Section: Dna Replicationmentioning

confidence: 99%

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Bacteriophage Ø29 protein p6: an architectural protein involved in genome organization, replication and control of transcription

González-Huici

Alcorlo

Salas

et al. 2004

J of Molecular Recognition

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Protein p6 of B. subtilis bacteriophage Ø29 binds to DNA forming a nucleoprotein complex in which the DNA wraps a protein core forming a right-handed superhelix, therefore restraining positive supercoiling and compacting the DNA. The protein does not specifically recognize a nucleotide sequence but rather a structural feature and it binds as a dimer through the minor groove. Protein p6 is in a monomer-dimer equilibrium that shifts to higher-order structures at a concentration of about 1 mM. These structures are probably present in vivo as the intracellular concentration of p6 is estimated to be in this range, and in fact the effective concentration should be still higher due to the macromolecular crowding. The p6 oligomers show an elongated shape compatible with a helical structure reminiscent of the superhelical DNA of the nucleoprotein complex, therefore it was proposed that protein p6 forms a scaffold on which the DNA folds. Since protein p6 is very abundant in infected cells, enough to bind the entire viral progeny, it was proposed to have an architectural role organizing and compacting the viral genome. It has been demonstrated that protein p6 binds in vivo to most, if not all, the Ø29 genome, although with different affinity, the highest one corresponding to the genome ends. Binding to plasmidic DNA was much lower, although it increased dramatically when the negative superhelicity was decreased. Hence, protein p6 binding specificity for Ø29 DNA is based on supercoiling, providing that the Ø29 genome, although topologically constrained, has a negative superhelicity lower than that of plasmid DNA. The formation of the nucleoprotein complex has functional implications in DNA replication and the control of transcription. It activates the initiation of replication that occurs at the genome ends for which the binding affinity is highest. It represses early transcription from promoter C2, and, together with protein p4, it represses transcription from promoters A2b and A2c and activates late transcription from promoter A3; therefore, protein p6 is involved in the early to late transcription switch.

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Section: Protein P6 Mutants Deficient In Self-associationmentioning

confidence: 91%

Section: Protein P6 Mutants Deficient In Self-associationmentioning

confidence: 99%

Section: Dna Replicationmentioning

confidence: 99%

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Bacteriophage Ø29 protein p6: an architectural protein involved in genome organization, replication and control of transcription

González-Huici

Alcorlo

Salas

et al. 2004

J of Molecular Recognition

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“…In addition to impaired dimer formation ability, mutations at these two residues showed reduced DNA binding affinity and they were affected in the initiation of 29 DNA replication. Thus, dimers seem to be the active form of 29 DBP for DNA binding (Abril et al, 2000).…”

Section: Replication Origins Of Tp Containing Genomesmentioning

confidence: 97%

Protein-Primed Replication of Bacteriophage ø29 DNA

de¹,

Salas²

2011

DNA Replication and Related Cellular Processes

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“…Structure-function work on p6 indicated that the N-terminal region of the protein plays a role in both DNA binding (Otero et al, 1990 ) and dimer formation (Abril et al, 2000 ). By site-directed mutagenesis, it could be disclosed that residues Ile8 and Val44 are directly involved in protein dimer formation (Abril et al, 2000 , 2002 ), whereas Lys2, Lys10 and, especially, Arg6, are essential for DNA binding in vitro and viral DNA synthesis in vivo (Bravo et al, 1994b ; Freire et al, 1994 ).…”

Section: A Histone-like Protein Encoded By Bacteriophage φ29mentioning

confidence: 99%

DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

Salas

Holguera

Redrejo-Rodríguez

et al. 2016

Front. Mol. Biosci.

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Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5′ ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3′–5′ exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the TP at the bacterial nucleoid, where viral DNA replication takes place. The biochemical properties of the Φ29 DBP and SSB and their function in the initiation and elongation of Φ29 DNA replication, respectively, will be described.

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Identification of Residues within Two Regions Involved in Self-association of Viral Histone-like Protein p6 from Phage Ø29

Cited by 6 publications

References 39 publications

Bacteriophage Ø29 protein p6: an architectural protein involved in genome organization, replication and control of transcription

Bacteriophage Ø29 protein p6: an architectural protein involved in genome organization, replication and control of transcription

Protein-Primed Replication of Bacteriophage ø29 DNA

DNA-Binding Proteins Essential for Protein-Primed Bacteriophage Φ29 DNA Replication

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